Fig. 1: Interactions with the cell wall slow the diffusion of Pal in the outer membrane of E. coli.

a Major components of the Tol system. The lipoprotein Pal, labelled with mCherry or PAmCherry (this work), non-covalently associates with the cell wall. Pal also binds to TolB through an interaction that is mutually exclusive of peptidoglycan binding. TolA spans the periplasm and is coupled to the PMF through interactions with two other inner membrane components, TolQ and TolR. The C-terminal domain of TolA interacts with the N-terminus of TolB (see below). Marked with an asterisk in TolA’s transmembrane helix is His22, which is essential for PMF coupling. b Pal-PAmCherry distribution in non-dividing (left-hand panel) and dividing (right-hand panel) cells. Panels show composite single-particle tracking data for 19,038 and 15,448 molecules in non-dividing and dividing cells, respectively. Data points represent the trajectory centroids of Pal-PAmCherry molecules normalised with respect to the cell. Pal has similarly low mobility in both cell types. Pal reorganises during cell division so that its concentration increases by ~55% at mid-cell relative to non-dividing cells. c Main panel, histogram showing apparent diffusion coefficients (D_app) for Pal-PAmCherry (red) and lipoylated-PAmCherry (grey), for clarity diffusion coefficients above 0.2 μm² s⁻¹ are not shown. Removing the peptidoglycan binding domain of Pal increases D_app at least fivefold (0.004 μm² s⁻¹, n = 22,794 tracks compared to 0.021 μm² s⁻¹, n = 30,640 tracks in SPT experiments) and mobility becomes less constrained. Insert: right, single-particle tracks of Pal-PAmCherry (red) and lipoylated-PAmCherry (grey) molecules normalised with respect to the cell, a selection of tracks with diffusion coefficients close to their respective medians are displayed. Insert: left, Box plots of Pal-PAmCherry and lipoylated-PAmCherry diffusion coefficients (outliers not shown) a two-sided Student’s t test with unequal variances indicated a p value of ≪0.0001. For Pal-PAmCherry the first, second and third quartiles are 0.001, 0.004 and 0.012 μm² s⁻¹, respectively. The whiskers represent the most extreme data points that lie within the third quartile +1.5× the interquartile range and the first quartile −1.5× the interquartile range: 0.029 and 1.5 × 10⁻⁷ μm² s⁻¹, respectively. For lipoylated-PAmCherry the first, second and third quartiles are 0.007, 0.021 and 0.048 μm² s⁻¹, respectively. The most extreme data points that lie within the third quartile +1.5× the interquartile range and the first quartile −1.5× the interquartile range are 0.109 and 1.5 × 10⁻⁶ μm² s⁻¹, respectively. Source data are provided as a Source Data file.